1. Field of the Invention
The present invention relates to endoglucanases and cellulase preparations containing the same, as well as methods of treating cellulose-containing fabrics, papers, pulps or animal feeds with the cellulase preparations.
2. Background Art
Treatment of cellulose-containing fabrics with cellulase is carried out to give the fabrics desired properties. For example, treatment with cellulase is carried out in the fiber industry in order to improve the touch and appearance of cellulose-containing fabrics or to give colored cellulose-containing fabrics an appearance of “stone-washed” material, i.e. partial color change (European Patent No. 307,564).
On the other hand, lyocell which is a regenerated cellulose fiber prepared from wood pulp-derived cellulose by an organic solvent spinning process has been attracting attention recently for its properties (such as high strength, water absorption) and the production process that causes less environmental pollution. However, since lyocell generates fuzz during its production, lyocell in that form is commercially evaluated rather low as a fabric product. Then, methods of removing the fuzz generating during production using cellulase have been proposed.
The term “cellulose-containing fabrics” used herein include fabrics prepared from cellulosic fiber materials such as natural cellulose (e.g., cotton, linen), regenerated cellulose (e.g., lyocell, rayon, polynosic, cupraammonium rayon); woven or knitted cloths from these fibers; and clothing items prepared by sewing these cloths. Further, those fabrics, cloths and sewn clothing items which comprise cellulosic fiber material(s) and other material(s) such as synthetic fiber, wool or silk, are also included in this term.
At present, cellulases derived from Trichoderma and Humicola (both are wood-rotting fungi) are mainly used in the treatment of cellulose-containing fabrics. These cellulases are mixtures of a plurality of cellulase components. Practical use of these cellulases has been hindered by the difficulty that a large quantity of cellulase preparation is needed in order to achieve a desired effect on a cellulose-containing fabrics.
The above-described drawback of these cellulose preparations is being improved by the development of preparations containing a large quantity of endoglucanases. For example, a number of endoglucanase-enriched cellulase preparations are disclosed in WO89/09259, WO91/17243, WO98/03640 and WO94/21801. In particular, WO91/17243 discloses that a Humicola-derived purified 43 kD endoglucanase component (EGV) exhibits jeans decoloring activity about 100-fold greater than that activity of conventional cellulase preparations which are mixtures of a plurality of cellulase components. WO98/03640 discloses that a Humicola-derived endoglucanase component NCE4 exhibits jeans decoloring activity and lyocell fuzz removal activity which are 25-fold and 100-hold greater than those activities of the conventional cellulase preparations, respectively. However, in order to remove strong fuzz generated in regenerated cellulose fabrics such as lyocell, cellulase preparations containing endoglucanase components of still higher activity are required to put them into practical use on an industrial scale.
Generally, the processing of cellulose-containing fabrics includes refining, bleaching, dyeing and mercerization; all of them are carried out under alkaline conditions. However, in the above-described conventional cellulose preparations containing a large quantity of endoglucanase, those derived from Trichoderma have an optimum pH in an acidic range and those derived from Humicola have an optimum pH in a neutral range. Thus, when these cellulase preparations are used, the cellulase treatment should be carried out separately from the above-mentioned fabrics processing steps, after pH adjustment by addition of buffers, etc.
Accordingly, if an endoglucanase component functioning under alkaline conditions is available, cellulase treatment can be carried out in the above-mentioned fabric processing steps. Thus, production steps can be shortened. As a result, it is believed that a great cost reduction can be achieved.
It is disclosed that Rhizopus-derived cellulases are capable of retaining their activity under alkaline conditions (Japanese Unexamined Patent Publications Nos. 60-226599, 64-40667, 64-26779 and 7-90300). In all of these disclosures, Rhizopus cultivation preparations are used for the purpose of providing detergents for use in the washing/rinsing of clothing. However, the activity of these Rhizopus cultivation preparations is extremely low and by far below the level required for practical use.
Highly active cellulase preparations are often provided as preparations containing a large quantity of endoglucanase as described above. For preparing such endoglucanase, methods are known in which an endoglucanase component of interest is recombinantly expressed in a host cell in a large quantity as described in WO91/17243, WO98/03667 and WO98/11139. Examples of preferable host cells in these methods include filamentous fungi belonging to Deuteromycotina, e.g., Aspergillus, Humicola, and Trichoderma. Considering enzyme production at an industrial level, these filamentous fungi belonging to Deuteromycotina will be extremely excellent hosts.
However, when a heterogeneous gene is expressed in these filamentous fungi belonging to Deuteromycotina, high expression is often hindered for reasons such as difference in codon usage. In particular, there has been no report of high expression of a gene derived from the genus Rhizopus belonging to Zygomycotina in the above-mentioned filamentous fungi belonging to Deuteromycotina. Thus, technology for high expression has been desired.
Under such circumstances, recently, a technology is being constructed which achieves high expression of a gene of interest in a host cell by optimizing the codons of the gene in conformity with the codon frequency in the host cell. The optimum use of codons for high expression of a gene of interest in a host may be presumed by examining the codon frequency in those genes expressed relatively abundantly in the host in natural environment. This is supported by the report of Lloyd et al. (Andrew T. Lloyd and Paul M. Sharp, 1991, Mol. Gen. Genet. 230, 288-294) concerning the codon frequency in Aspergillus nidulans, a filamentous fungus belonging to Deuteromycotina. However, even if information on appropriate codon use has been obtained from known DNA sequences, that information does not necessarily result in the realization of high expression of a gene of interest immediately. Especially, in filamentous fungi, which are complex to control, selection of a single sequence most suitable for expression from a number of sequences having suitable codon use has been required.